ABSTRACT
AIMS: To assess the applicability of sequence characterized amplified region (SCAR) markers obtained from BOX, ERIC and RAPD fragments to design primers for real-time PCR quantification of the phytostimulatory maize inoculants Azospirillum brasilense UAP-154 and CFN-535 in the rhizosphere. METHODS AND RESULTS: Primers were designed based on strain-specific SCAR markers and were screened for successful amplification of target strain and absence of cross-reaction with other Azospirillum strains. The specificity of primers thus selected was verified under real-time PCR conditions using genomic DNA from strain collection and DNA from rhizosphere samples. The detection limit was 60 fg DNA with pure cultures and 4 x 10(3) (for UAP-154) and 4 x 10(4) CFU g(-1) (for CFN-535) in the maize rhizosphere. Inoculant quantification was effective from 10(4) to 10(8) CFU g(-1) soil. CONCLUSION: BOX-based SCAR markers were useful to find primers for strain-specific real-time PCR quantification of each A. brasilense inoculant in the maize rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: Effective root colonization is a prerequisite for successful Azospirillum phytostimulation, but cultivation-independent monitoring methods were lacking. The real-time PCR methods developed here will help understand the effect of environmental conditions on root colonization and phytostimulation by A. brasilense UAP-154 and CFN-535.
Subject(s)
Azospirillum brasilense/genetics , DNA Primers/chemistry , Polymerase Chain Reaction/methods , Zea mays/microbiology , Azospirillum brasilense/classification , Azospirillum brasilense/growth & development , DNA Fingerprinting , DNA, Bacterial/chemistry , Genetic Markers , Plant Roots/microbiology , Random Amplified Polymorphic DNA Technique , Rhizosphere , Soil MicrobiologyABSTRACT
Assembly of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex is an essential step for neurotransmitter release in synapses. The presynaptic plasma membrane associated proteins (t-SNAREs), SNAP-25 (synaptosome-associated protein of 25,000 Da) and syntaxin 1A may form an intermediate complex that later binds to vesicle-associated membrane protein 2 (VAMP2). Using spin labeling electron paramagnetic resonance (EPR), we found that the two t-SNARE proteins assemble into a parallel four-helix bundle that consists of two identical syntaxin 1A components and the N-terminal and C-terminal domains of SNAP-25. Although the structure is generally similar to that of the final SNARE complex, the middle region of the helical bundle appears more flexible in the t-SNARE complex. Such flexibility might facilitate interactions between VAMP2 and the t-SNARE complex.
Subject(s)
Membrane Proteins/chemistry , Neurons/chemistry , Vesicular Transport Proteins , Electron Spin Resonance Spectroscopy , Macromolecular Substances , Membrane Proteins/metabolism , Models, Molecular , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Pliability , Protein Structure, Secondary , Qa-SNARE Proteins , SNARE Proteins , Spin Labels , Synaptosomal-Associated Protein 25 , TemperatureABSTRACT
The heterotrimeric synaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consisting of the synaptic vesicle-associated membrane protein 2 (VAMP2) and presynaptic plasma membrane proteins SNAP-25 (synaptosome-associated protein of 25,000 Mr) and syntaxin 1A, is a critical component of the exocytotic machinery. We have used spin labeling electron paramagnetic resonance spectroscopy to investigate the structural organization of this complex, particularly the two predicted helical domains contributed by SNAP-25. Our results indicate that the N- and C-terminal domains of SNAP-25 are parallel to each other and to the C-terminal domain of syntaxin 1A. Based on these findings, we propose a parallel four-stranded coiled coil model for the structure of the synaptic SNARE complex.
Subject(s)
Membrane Proteins/chemistry , Nerve Tissue Proteins/chemistry , Synapses/chemistry , Vesicular Transport Proteins , Animals , Antigens, Surface/chemistry , Antigens, Surface/genetics , Cyclic N-Oxides , Electron Spin Resonance Spectroscopy , In Vitro Techniques , Macromolecular Substances , Membrane Proteins/genetics , Mesylates , Models, Molecular , Mutagenesis , Nerve Tissue Proteins/genetics , Protein Conformation , Protein Structure, Secondary , R-SNARE Proteins , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , SNARE Proteins , Spin Labels , Synaptosomal-Associated Protein 25 , Syntaxin 1ABSTRACT
A stable ternary complex formed with vesicle-associated membrane protein 2 (VAMP2) and plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) is proposed to function in synaptic vesicle exocytosis. To analyze the structural characteristics of this synaptic protein complex, recombinant binary (syntaxin 1A.SNAP-25), recombinant ternary, and native ternary complexes were subjected to limited trypsin proteolysis. The protected fragments, defined by amino-terminal sequencing and mass spectrometry, included a carboxyl-terminal region of syntaxin 1A, the cytoplasmic domain of VAMP2, and amino- and carboxyl-terminal regions of SNAP-25. Furthermore, separate amino- and carboxyl-terminal fragments of SNAP-25, when combined with VAMP2 and syntaxin 1A, were sufficient for stable complex assembly. Analysis of ternary complexes formed with full-length proteins revealed that the carboxyl-terminal transmembrane anchors of both syntaxin 1A and VAMP2 were protected from trypsin digestion. Moreover, the stability of ternary complexes was increased by inclusion of these transmembrane domains. These results suggest that the transmembrane domains of VAMP2 and syntaxin 1A contribute to complex assembly and stability and that amino- and carboxyl-terminal regions of SNAP-25 may function as independent domains.
Subject(s)
Endopeptidases/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Animals , Biopolymers , Hydrolysis , Membrane Proteins/chemistry , Nerve Tissue Proteins/chemistry , Protein Conformation , Qa-SNARE Proteins , R-SNARE Proteins , Rats , Sodium Dodecyl Sulfate , Synaptosomal-Associated Protein 25 , Syntaxin 1ABSTRACT
One lot of meprobamate raw material and 28 lots of tablets were examined for impurities by TLC. All lots contained di-(2-methyl-2-propyl-3-carbamoyloxypropyl) carbonate (V) at levels that ranged between 0.1 and 1.0% of the total drug content. Nine lots also contained low levels of a second impurity (approximately 0.1%), and one of these lots contained a third impurity (approximately 0.1%), neither of which was identified. Estimates of the unidentified impurities were based on the assumption of a TLC response with furfural-hydrochloric acid spray equivalent to that of meprobamate. Compound V was identified by mass spectrometry and PMR and IR spectroscopy and by comparison of the TLC Rf value to that of a synthesized sample of V.
Subject(s)
Meprobamate/analysis , Chromatography, Thin Layer , Drug Contamination/analysisABSTRACT
Two previously reported but unidentified phenylburazone degradation products were isolated from a tablet that was stored at 60 degrees for 203 days. The compounds, alpha-(N-phenylcarbamoyl)-N-caproylhydrazobenzene and alpha-hydroxy-alpha-(N-phenylcarbamoyl)-N-caproylhydrazobenzene, were isolated by chromatography, identified by mass and NMR spectrometry, and synthesized by the reaction of aniline with phenylbutazone or its hydroxy analog, respectively.
Subject(s)
Phenylbutazone/analysis , Chemical Phenomena , Chemistry , Magnetic Resonance Spectroscopy , Mass Spectrometry , Phenylbutazone/standards , Tablets/standardsABSTRACT
Two lots of trihexyphenidyl hydrochloride raw material, one lot of elixir, and 10 lots of tablets were examined for impurities by TLC. Impurities found were 1-phenyl-2-propenone, 3-piperidinopropiophenone, and 3-aminopropiophenone. Not all impurities were present in all lots, and none exceeded 1.9% of the label drug claim. Impurities were identified by mass spectrometry and by comparison of TLC Rf values and GLC retention times to those of synthesized specimens of the impurities.
